Micro-Sensors to Study Electrical and Mechanical Coupling of Injured Myocardium

摘要 心力衰竭仍然是美国发病率和死亡率的主要原因，困扰着近500万人。 最近，成年斑马鱼（Danio rerio）已被用于模拟不同类型的心力衰竭，并搜索 通过诱变筛选获得遗传修饰剂。然而，斑马鱼心脏的小尺寸阻碍了精确的 遗传修饰后的电气和机械评估。在上一个融资周期，我们 将柔性微电极阵列与高频超声换能器集成，以证明早期 再生的心肌细胞缺乏整合到受损心脏所需的电表型。我们进一步 表明房室瓣上的压力梯度大于 心室冷冻损伤后的心室小叶瓣。然而，最初的上升和随后的正常化， 心室被动（E）和主动（A）充盈波（E/A比）表明舒张功能恢复。未来 我们将联合收割机将我们的微传感能力与新型心肌病遗传模型相结合， 阐明化疗诱导的损伤后的机电耦合和 心肌病我们的多学科团队建立了多柔比星（Dox）诱导的成年斑马鱼模型， 心肌病（CM）作为一种保守的脊椎动物模型，以研究心肌损伤和再生， 对靶向ErbB 2（HER 2）/NEU的乳腺癌化疗的反应。我们的团队进一步发展了 CM的三种鼠遗传模型;即，bag 3敲除（KO）、mBAG 3过表达（OE）和Imna KO。 我们进一步开发了一种正向遗传方法来识别Dox诱导的CM的遗传修饰剂。一个试点 筛选了>500个基因断裂转座子（GBT）突变体，鉴定了四个GBT系，其中GBT 419/rxraa （类维生素A X受体α a）类似于mTOR以改善Dox诱导的CM后的斑马鱼存活。我们的目标 是将微传感器与先进的成像技术相结合，以研究电传导和机械功能， Dox诱导的心肌损伤模型和3种CM遗传模型。我们的假设是， 修饰剂如GBT 419/rxraa促进了Dox诱导的和遗传模型中的机电耦合， CM恢复收缩功能。为了验证我们的假设，我们有三个目标：在目标1中，我们将确定 在我们的Dox诱导和遗传模型中的电传导。在目标2中，我们将演示机械 在我们的Dox诱导和遗传模型中发挥作用。在目标3中，我们将评估机电耦合 用CM修饰基因处理后。总的来说，这些目标将提供新的见解， 利用正向遗传学发现心肌病中的机电耦合， 恢复心脏功能。

ABSTRACT Heart failure remains the leading cause of morbidity and mortality in the US, afflicting nearly 5 million people. Recently, adult Zebrafish (Danio rerio) have been utilized to model different types of heart failure, and to search for genetic modifiers via mutagenesis screening. However, the small size of the zebrafish heart hinders precise electrical and mechanical assessments following genetic modifications. During the previous funding cycle, we integrated a flexible micro-electrode array with high-frequency ultrasonic transducers to demonstrate that early regenerating cardiomyocytes lack the electrical phenotypes needed to integrate into injured hearts. We further showed that the pressure gradient across the atrioventricular valve is greater than that across the ventriculobulbar valve following ventricular cryo-injury. However, the initial rise and subsequent normalization of ventricular passive (E) and active (A) filling waves (E/A ratios) indicate recovery of diastolic function. In the next funding cycle, we will combine our micro-sensing capacity with novel genetic models of cardiomyopathy to elucidate electromechanical coupling following chemotherapy-induced injury and genetic models of cardiomyopathy. Our multi-disciplinary team established an adult zebrafish model of doxorubicin (Dox)-induced cardiomyopathy (CM) as a conserved vertebrate model to investigate myocardial injury and regeneration in response to the breast cancer chemotherapy targeting ErbB2 (HER2)/NEU. Our team has further developed three murine genetic models of CM; namely, bag3 knockout (KO), mBAG3 overexpression (OE), and Imna KO. We have further developed a forward-genetic approach to identify genetic modifiers of Dox-induced CM. A pilot screen of >500 gene-breaking transposon (GBT) mutants has identified four GBT lines, of which GBT419/rxraa (retinoid X receptor alpha a) resembles mTOR to improve zebrafish survival following Dox-induced CM. Our goal is to integrate micro-sensors with advanced imaging to study electrical conduction and mechanical function of the injured myocardium in response to Dox-induced and 3 genetic models of CM. Our hypothesis is that genetic modifiers such as GBT419/rxraa promotes electromechanical coupling in Dox-induced and genetic models of CM to restore contractile function. To test our hypothesis, we have three aims: In Aim 1, we will determine electrical conduction in our Dox-induced and genetic models. In Aim 2, we will demonstrate mechanical function in our Dox-induced and genetic models. In Aim 3, we will assess electromechanical coupling following treatments with CM modifying genes. Overall, these aims will provide new insights into electromechanical coupling in cardiomyopathy using forward-genetics to discover therapeutic modifiers capable of restoring heart function.